Obesity is a medical problem of increasing concern. An individual is defined as being clinically obese if they have a body mass index greater than 30 kg/m2. In 1962, research statistics showed that the percentage of obesity in America's population was at 13%. By 1980 it has risen to 15%, by 1994 to 23%, and by the year 2000 the number of individuals in America categorized as being clinically obese had reached an unprecedented 31%. As a result, the health care service reports high morbidity and mortality from weight-related conditions such as cardiovascular disease and certain forms of cancer, and has seen a dramatic increase in type 2 diabetes mellitus. The U.S. Surgeon General report declared that obesity is responsible for 300,000 deaths every year. Understanding the molecular mechanisms underlying obesity has therefore become the object of an intense research effort.
Adipocytes typically develop from mesodermal stem cells, which can also differentiate into other mesenchymal cells such as muscle cells and osteoblasts. It has been shown that insulin, insulin-like growth factors, growth hormone, glucocorticoids, and catecholamines affect fat cell proliferation and differentiation in vivo and in vitro. See, for example, Gregoire et al., Physiol. Rev. 78, p. 783-809 (1998). In addition, metalloproteases can also be involved in adipogenesis, where they play a significant role in extracellular matrix (ECM) remodeling. See, for example, Lilla et al., Am J. Pathol. 160, p. 1551-4 (2002).
ADAM 12 is a member of the family of proteins known as ADAMs (a disintegrin and metalloprotease). The ADAMs constitute a large family of multidomain membrane-anchored proteins that have been implicated in a number of biological activities, including extracellular-matrix remodeling, myogenesis, and adipogenesis. ADAM 12 exists in two forms: a membrane-bound long form, ADAM 12L, and an alternatively spliced secreted short form, ADAM 12S, which includes a prodomain and metalloprotease, disintegrin, cysteine-rich, and epidermal growth factor (EGF)-like domains. At the COOH-terminus, ADAM 12L contains a transmembrane domain and a cytoplasmic tail, whereas ADAM 12S is not membrane-anchored and contains a unique stretch of 33 amino acids.
Previous research using ADAM 12S transgenic mice under the transcriptional regulation of a muscle creatine kinase (MCK) promoter exhibit adipogenesis among muscle fibers as evidenced by Oil Red O staining and PPARγ positive cells (Kawaguchi et al., Am. J. Path., 160, p. 1895-1903 (2002)). Results from these transgenic mice suggest that ADAM 12S stimulated the formation of white adipose tissue. The authors indicated that both the membrane anchored ADAM 12L and the secreted forms of ADAM 12 (ADAM 12S) could stimulate adipogenesis.
Heparin-binding EGF-like growth factor (HB-EGF) is a membrane bound protein that is proteolytically processed by the soluble form of ADAM 12. HB-EGF has been implicated in a wide variety of disorders, including tumor growth, heart disease, and obesity. ADAM 12S stimulates ectodomain shedding of pro-HB-EGF, releasing a mature, soluble ligand (sHB-EGF) and a carboxyl-terminal fragment (HB-EGF-C) consisting of the transmembrane and cytoplasmic domains. Higashiyama et al., Biochim Biophys Acta., 1751, p. 110-7 (2005).
Mature, soluble HB-EGF and HB-EGF C are both known to stimulate cellular proliferation. Zhou et al., Cell Prolif., 40, p. 213-30 (2007). It was therefore the expectation that cells expressing both HB-EGF and ADAM 12 would exhibit significant cell proliferation as a result of proHB-EGF being proteolytically cleaved by ADAM 12S to provide soluble HB-EGF and HB-EGF C.